AU594804B2 - Small molecular weight antigen of plasmodium falciparum - Google Patents

Description

AU-Al-76982/87 P T WORLD INTELLECTUAL PROPERTY ORGANIZTIO, PCT -International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PA TV OPEAT ATY CT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 00597 C07K 7/06, 15/12, C12N 5/00 Al C12N 15/00, A61K 39/015, 39/395 (43) International Publication Date: 28 January 1988 (28.01.88) (21) International Application Number: PCT/AU87/00226 194 Arthur Terrace, Bardon, QLD 4065 GEYS- EN, Hendrik, Mario [AU/AU]; 10 Janden Close, (22) International Filing Date: 17 July 1987 (17.07.87) Knoxfield, VIC 3180 SAUL, Allan, James [AU/ AU]; 14 Dajarra Street, The Gap, QLD 4061 (AU).

(31) Priority Application Numbers: PH 6983 PI 1046 (74) Agents: SLATTERY, John, Michael et al.; Davies Collison, 1 Little Collins Street, Melbourne, VIC (32) Priority Dates: 17 July 1986 (17.07.86) 3000 (AU).

24 March 1987 (24.03.87) (81) Designated States: AT (European patent), AU, BE (Eu- (33) Priority Country: AU ropean patent), CH (European patent), DE (European patent), DK, FR (European patent), GB, GB (71) Applicant (for all designated States except US): SARA- (European patent), IT (European patent), JP, KR, LU MANE PTY. LTD. [AU/AU]; The Walter atid ELiza (European patent), NL (European patent), SE (Euro- Hall Institute of Medical Research, Royal Parade, pean patent), US.

Parkville, VIC 3052 (AU).

Published 3 (72) Inventors; and With international search report.

Inventors/Applicants (for US only) KARA, Ursula, An-- l na, Kate [DE/AU]; 2/11 Munro Street, Kelvin Grove, 7 AR S QLD 4059 STENZEL, Deborah, Joan [AU/ A-..JP 7 MAR 1988 AU]; 3 Tarcoola Avenue, Ferny Hills, QLD 4055- BUSHELL, Gillian, Robin [AU/AU]; AUSTRALIAN FEB 1988 o PATENT OFFICE

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t a r z (54) Title: SMALL MOLECULAR WEIGHT ANTIGEN OF PLASMODIUM FALCIPARUMT cii1 (57) Abstract A small molecular weight antigen of the asexual blood stages of Plasmodiumfalciparum which is characterized by: having an apparent molecular weight in the range of approximately 15 kD to 19 kD; (ii) not showing significant glycosylation by galactose or glucosamine labelling, but being acylated by myristic acid; (iii) being associated with the parasitophorous vacuole membrane and with inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell; and (iv) being recognised by monoclonal antibodies against the asexual blood stages of P. falciparum which inhibit parasite growth in vitro; or an antigenic fragment thereof. A monoc!ial antibody specific for this antigen, and a hybrid cell line which produces this antibody is also disclosed.

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ii i 1 2:-i 1 i d t 1 a,*K tJ ;1 WO 88/00597 PCT/AU87/00226 '1 SMALL MOLECULAR WEIGHT ANTIGEN OF PLASMODIUM FALCIPARUM This invention relates to the identification of antigens of the asexual blood stages of Plasmodium falciparum which are capable of generating antibodies which are able to inhibit the growth of the parasite, and to the use of these antigens and antibodies in immunizing, diagnostic and treatment methods.

SAs is well known, malaria, a widespread disease of man and {1 other vertebrates, is caused by a unicellular protozoan of the genus 1 Plasmodium and is transmitted by the bite of female Anopheline mosquitoes. Among the four species which infect man, Plasmodium falciparum is by far the most lethal. The pathological consequences of malaria, characterised by febrile paroxysms, anemia, splenomegaly and debilitation, are due to the development and proliferation of asexual blood stages in the vertebrate host.

For most of its life cycle in man, Plasmodium falciparum is 1 t 1 ;i r Lt:ioi- L i :i WO 88/0097 PCT/AU87/00226 _z 'h J *1 located inside its host cell with the exception of two stages of a brief extracellular existence, the sporozoite and the free merozoite.

A number of plasmodial antigens have been identified which appear to be associated with membranes of the parasite or of the infected host cell. Considerable attention has been directed to the sporozoite surface membrane the merozoite surface membrane and the plasma membrane of the parasitized erythrocyte One of the intriguing events of the invasion process of the host erythrocyte by the merozoite is the formation of the parasitophorous vacuole. The membrane surrounding the vacuole is a simple membrane bilayer, almost devoid of intramembranous particles (IMPs) at the early stage of infection It has been suggested that components both of the red blood cell membrane and of the invading parasite (merozoite coat and/or rhoptry material) make up the parasitophorous. vacuole membrane In the course of schizogony, morphological changes occur in the vacuole membrane such that proteins presumably synthesized by the parrasite become inserted therein. It has long been proposed that the parasitophorous vacuole contains antigenic material. Scaife et.al.

have suggested that the Ag5.1 is located within or close to the vacuole membrane, while it appears that S antigens accumulate within the vacuole The quest for a vaccine against malaria requires the investigation of malarial antigens capable of eliciting a protective response. Two approaches have been used to identify such antigens.

One is to compare the antigens recognised by antibodies in sera from individuals exposed to malaria, and correlate particular antigens recognised with the degree of protective immunity exhibited by the sera. A variation on this approach involves the use of human immune sera in the characterisation of antigens cloned in E.coli using recombinant-DNA techniques. The second approach is to use hybridoma technology to produce murine monoclonal antibodies (MAB) which inhibit the growth of malaria in vitro then to identify the corresponding antigen. This approach has led to the production of inhibitory antibodies directed against the sporozoites and asexual and sexual I WO 88/00597 PCT/AU87/00226 3 blood forms of several species ("Recent Progress in the Development of Malaria vaccines: Memorandum from WHO Meeting". Bulletin of the World Health Organistion 62 7150727 (1984)).

Holder Freeman (25) have been able to use single antigens of Plasmodium yoelii identified by monoclonal antibodies as vaccines in mice to elicit immunity. In this case the antigen used was purified from infected cells. While it may be possible to prepare sufficient antigen for vaccine trials from cells infected with the human species of malaria, large scale production of vaccines will probably requirs recombinant DNA technology. This places additional constraints upon the antigen, e.g. it would be difficult to prepare a vaccine based upon a polysaccharide antigen by this route. Thus, protein antigens are more attractive from this viewpoint.

Monoclonal antibodies directed against defined parasitic antigens have been shown to inhibit the development of erythrocytic stages of Plasmodium falciparum in vitro, Perrin et.al. and to -prevent release of the merozoites or prevent invasion of the red blood cells by merozoites (in accordance with the present invention).

A method for the identification of antigens of the asexual blood stages of the Plasmodium falciparum parasite which can be used' to generate antibodies which are able to inhibit the growth of the Sparasite comprises: growing Plasmodium falciparum isolates in vitro thereby producing erythrocytes infected with the Plasmodium falciparum parasite; purifying these erythrocytes; immunising mice with the purified erythrocytes; d) fusing spleen cells from the immunised mice with a mouse myeloma cell line to produce hybridomas; testing the monoclonal antibodies produced by the hybridomas ,i to identify those hybridoma lines which secrete monoclonal antibodies against the Plasmodium falciparum parasite; characterising the monoclonal antiparasite antibodies; S(g) preparing mouse ascites fluid by injecting mice p 3

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WO 88/00597 PCT/AU87/00226 intraperitoneally with hybridoma cells secreting monoclonal antiparasite antibodies; extracting and purifying monoclonal antibodies from these ascites fluids; adding the purified antibodies to Plasmodium falciparum cultures in vitro and subsequently examining the cultures for a decrease in the multiplication rate of the parasites; characterising the antigen recognised by each inhibitory monoclonal antibody; identifying the cellular distribution of the antigen and the stage of parasite maturation during which it is present.

According to the present invention, there is provided a small molecular weight antigen of the asexual blood stages of Plasmodium falciparum which is characterised by: having an apparent molecular weight in the range of approximately 15 kD and 19 kD; (ii) not showing significant glycosylation by galactose or glucosamine labelling, but being acylated by myristic acid; (iii) being associated with the parasitophorous vacuole membrane and with inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell; and S(iv) being recognised by monoclonal antibodies against the asexual Sblood stages of P.falciparum which inhibit parasite growth in vitro; 25 or an antigenic fragment thereof.

Preferably, the antigen is the antigen QF116 described in detail herein, or an antigenic fragment thereof.

As the small molecular weight antigen described herein is Sclosely associated with both the parasitophorous vacuole membrane and vesicles as well as cytoplasmic clefts, it could conceivably be Sinvolved in the traffic of proteins.

In another aspect, this invention provides a hybrid cell line which produces an antibody which is specific for the small molecular weight antigen described above, as well as an antibody produced by *0 j 1 i :fi 1 1 1 1 1 1 11 1 1 1 1 i i i WO 88/00S97 PCT/AU87/00226 such a hybrid cell line.

Preferably, the antibody is an IgGl, monoclonal antibody, Smore preferably it is the monoclonal antibody 8E7/55 described in detail herein. Monoclonal antibody 8E7/55 is produced by the hybrid cell line deposited at the European Collection of Animal Cell Cultures, Porton Down, Salisbury, England, on July 10, 1987 under No. 87071009 Monoclonal antibody 8E7/55 specifically recognises antigen QF116 as described below.

Monoclonal antibodies of the present invention are characterised in that they are inhibitory, in that they either prevent release of merozoites into the blood stream of an infected individual, prevent these merczoites from invading the red blood cells of such an individual, and prevent intracellular development of parasites in the red blood cells of such an individual.

In another aspect, the present invention relates to an antigen of P.falciparum which is characterised by inclusion of the amino acid sequence Asn-Leu-Val-Ser-Glu-Pro (NLVSGP), or an antigenically active related sequence derived therefrom.

The invention further provides a method of inhibiting the development of asexual blood stages of Plasmodium falciparum which 1: metlod ctinprises exposing the blood stages of the parasite to antibodies according to the present invention.

SThe invention also provides a method for passively immunising a host against Plasmodium falciparum which method comprises administering to the host antibodies according to the present invention.

SThe invention also provides a method for actively immunising a ho'ct against Plasmodium falciparum which method comprises r administering to the host an antigen according to the present invention, or an antigenic fragment thereof. Preferably the antigen or fragment is one recognised by at least one monoclonal antibody I according to the invention. More preferably the antigen or fragment is recognised by 8E7/55. Most preferably the antigen is QF116 or an r :FnX;i up: WO :88/0597 PCT/AU87/00226 antigenic fragment thereof.

The invention provides a vaccine comprising an antigen according to the present invention, preferably antigen QF116, or an antigenic fragment thereof, a pharmaceutically acceptable carrier or diluent and optionally an adjuvant. The invention also provides a passive vaccine comprising antibodies according to the invention, preferably monoclonal antibody 8E7/55, and a pharmaceutically acceptable carrier or diluent.

The invention also provides a reagent for purification of Plasmodium falciparum antigens comprising an antibody according to the invention.

In addition the invention provides a diagnostic reagent for detection of Plasmodium falciparum or antigens derived therefrom comprising an antibody according to the invention.

As a result of applying the general techniques described above, a range of monoclonal antibodies have been identified as able to inhibit the growth of malaria in vitro, and one of these inhibitory monoclonal antibodies have been investigated in greater detail as outlined below. The immunisation regime and fusion leading to the production of monoclonal antibody 8E7/55 were performed essentially as described in Schofield et.al. (14) and Saul et.al. A mouse was immunised with 107 red cells infected with mature schizonts of the PNG isolate FCQ-27/PNG. These cells were purified from culture by the method of Saul, et.al.(12). The cells were injected intraperitoneally with 0.1 ml Freund's complete adjuvant. The mice were boosted 4 weeks later by an intravenous injection of 5 x 10 schizont infected red cells in saline. A similar boost was given after a further 2 weeks.

Four days after the last injection, the spleen cells were harvested and fused.

Monoclonal antibodies were purified by affinity chromatography on Protein A Sepharose (Pharmacia) and were tested for inhibition of invasion using a similar method to that described in Schofield(14). Briefly, purified monoclonal antibodies in 1640 medium were added to synchronised parasites at ring stage and

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/4 1 I ^1 J 4~..44/ 9"' l v 1 "*7 WO 88/00597 PCT/AU87/00226 7 incubation of the parasites continued to allow reinvasion. [3 S] Methionine (3-4pCi) or H]-hypoxanthine (3-4.ICi) was added to each well and growth of the parasite allowed to proceed to trophozoite stage. The samples were harvested, protein precipitated with ice cold 10% trichloro-acetic acid onto glass fibre filters and the filters Swere washed and the radioactivity remaining was measured in a scintillation counter. The class and subclass of the monoclonal antibodies were determined by immunodiffusion in agar using rabbit antisera specific for mouse IgM, IgGl, IgG2a, IgG2b and IgG3.

Isoelectric points were determined by isoelectric focussing of hybridomd culture supernatant in thin layer agarose gels. Following the focussing, the gels were fixed, washed in phosphate buffered saline, soaked in 1I-labelled antibody to mouse Ig, washed, dried and the position of the radioactive label determined by autoradiography.

Preliminary characterisation of the antigen (antigen QF116) Srecognised by the monoclonal antibody 8E7/55 as described in detail below revealed the following: SSize: The antigen is detectable on Western blots of FCQ-27/PNG with the monoclonal antibody described givina a band of 1.9 kn. On immunoprecipitation a major band at 19 kD is detected; occasionally weak bands of higher molecular weight are seen. Immunoprecipitation 3 of antigen from parasites biosynthetically labelled with H-myristic acid also gives a 19 kD band, consistent with acylation of the protein.

Cellular location and stage specificity: j The antigen is detectable in all blood stages. The fluorescence appears over the whole asexual parasite, sometimes localised outside the parasite but inside the host red cell. On immunoelectron /icroscopy at the schizont stage the antigen is 1.

associated with the parasitophorous vacuole membrane and with vesicles.

Monoclonal: The antigen is recognised by the monoclonal antibody 8E7/55 which is an IgG 1 class antibody with a pI of 7.05 on isoelectric Sfocussing.

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WO 88/00597 PCT/AU87/00226 Inhibition: The monoclonal 8E7/55 gives 30-55% inhibition at 0.06-0.6 .3 mg/mL H]-hypoxanthine assay).

Further details of the characterisation of antigen QF116 and monoclonal antibody 8E7/55 are set out in the following Example.

EXAMPLE

MATERIALS AND METHODS Parasite cultures. Parasite strains FCQ-27 from Papua New Guinea [10) and FCR- 3K+ from West Africa were grown in synchronous cultures as previously described [111. Parasite infected erythrocytes at different stages of development were harvested on Percoll gradients [12].

Monoclonal antibody. The 8E7/55 hybridoma line used in this study was derived from mice immunized with schizonts of the FCQ-27 PNG strain and was cloned by limiting dilution E13]. The monoclonal antibody of the IgG1 class used for immunoelectron microscopic studies was purified from ascites fluid by affinity chromatography on Protein A-Sepharose (Pharmacia Chemicals).

Inhibition of parasite growth in vitro. Affinity purified antibody was added in varying concentrations to quadruplicate cultures of synchronised FCQ-27 in 96well plates. The wells contained 45 pl RPHI 1640/TES. 10% pooled human serum, with a 5' haematocrit and 1-2% schizont parasitemia, and 5 1l of test antibody.

Control wells were identical in all respects except that they contained either affinity purified normal mouse IgG or medium alone. The cultures were incubated overnight at 37 C. Following merozoite release and subsequent invasion 100 p 1 medium was added containing 10% pooled human serum and supplemented with 30 pCi mi H]hypoxanthine (Amersham). The parasites were allowed to develop for 40 h to the mature schizont stage, and harvested by pipetting well contents onto Whatman glass fibre discs which were washed in trichloroacetic acid (TCA) and ethanol. The dry discs were immersed in 3 ml toluene scintillant and counted in a Packard Scintillation Counter.

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%ijl r i iI i j i: r Irt~ ,i a o4 -memmm a) i:; WO 88/00597 9 PCT/AU87/00226 Affinity purification of antigen, MAb 8E7/55 was covalently linked to cyanogen bromide-activated Sepharose (Pharmacia) according to the manufacturer's instructions, and a I% Triton X-114 extract of FCQ-27 was passed through the column. After extensive washings the bound antigen was eluted with 0.2 M glycine, pH 2.8.

Immunofluorescence microscopy. IFAT was performed on fixed and unfixed parasites. For the former, thin films of parasitized blood were methanol-fixed and incubated with monoclonal antibody and FITC-labeled, affinity purified goatanti-mouse IyG as described elsewhere (143. IFA on unfixed parasites was performed using 200 gl of parasite culture at a defined stage. Schizont infected erythroctes were treated with anti-red blood cell antibody (CSL) plus complement for 60 minutes at 37 C. A two hour incubation of parasites with monoclonal antibody at 37*C was folloued by a one hour incubation with FITClabeled secondary antibody at 37 C. Parasites were gently but extensively washed with medium after each incubation step. Parasites were mounted on microscope slides and examined by fluorescence microscopy.

Immunoelectron microscopy. Immunoelectron microscopy was carried out on thin sectioned parasites at the ring or schizont stage using colloidal gold as an electron-dense marker. Samples of parasite culture were fixed with glutaraldehyde fo e 10 minutes at room temperature. Cells were washed three times in 0.1 M cacodylate buffer, dehydrated in 50 ethanol followed by ethanol, infiltrated with LR-White resin (London Resin Co. Ltd.) and polymerize at 50' C for 24 hours. Thin sections were mounted on nickel grids, rinsed with water, blocked with 5% BSA, 0,05% Tween 20 in 20 mH Tris-buffer. and incubated l r *with monoclonal antibody for bd minutes. One hour incubations with secondary antibody (rabbit anti-mouse) and tertiary antibody (goat anti-rabbit, colloidal gold conjugated, 10-15mm; Janssen Pharmaceutica, Belgium) followed. Sections were washed extensively after each incubation step, before being contrasted with lead citrate and uranyl acetate and viewed with a Philips EM400 electronmicroscope.

-a aio i-M ta l 1 w "s t u' iF s i' T~ r '3 |S I~ w n 1> WO 88/00597 WO 8890597PCT/A U87/00226 Biosynthetic labeling studies. Malarial proteins were radiolabeled by uptake of -1 _3 14 pCi ml R2 Hkqlycine. 25 pCi ml 1 Clleucine or 100 gCi ml- I S]methionine in RPIII medium. Glycoproteins were labeled by incorporation of uC Ii ml 1 D-C 6 3Hlgluco Isamine hydrochloride or 25 gCi ml1 1 14Clgalactose.

radiochemicals were purchased from Amersham Radiochemical Centre. Metabolic labeling with fatty acid was performed in RPMI medium using 50 uCi m1 1 9,10- 3 H~myristic acid coupled to B3SA. Synchronous cultures at ring stage were labelled for uo to 20 hours depending ona the precursor used with the exception Iof 3 S] methio'nine which was added to schizont stage cultures. Parasitized cells were washed and parasites extracted by saponi,,n treatment Protease inhibitors (5 mg ml-1 each of pepstatin, leupeptin, chymostatin and antipain) were added and detergent solubilized proteins were used for immunoprecipitation.

Imnunoprecipitaton. Aliquots of total parasite or Triton X-114 extracts from radiolabeled schizonts were incubated with ascites fluid of monoclonal antibody overnight at 4 0 C. Immune complexes were then precipitated with rabbit antimouse IgG/Protein A-Sepharose. The precipitate was washed in PBS/Triton and the immune complex was eluted with Laemmli sample'buffer as described for use in SDS-PAGE [16].

SDS-PAGE and autoradiocqraphy. Radiolabeled proteins were ''alysed on 121 -or SDS-polyacrylamide gels according to the Laeminli procedure Following electrophoresis gels were fluorographed with Amplify (Amersham) for 30 minutes, dried and autoradiographed on Kodak XAR 5 film at -70 0

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I: 6 WO 88/00597 PCT/AU87/0022 Immunoblotting. After separation on SDS-PAGE parasite proteins were electrophorecically transferred onto nitrocellulose paper, immunoreacted with MAb 8E7/55 and 125-labeled goat anti-mouse antibody, following the Western blotting procedure of Towbin et.al. Human serum used in immunoblotting was affinity-purified on the recombinant protein expressed by Ag61 [27].

Scanning. Scanning for antibody-reactive peptides using the predicted sequence Cof ,he polypeptide coded for by the insert of Ag61 was performed as described by Geysen et.al. Synthetic peptides were coupled via a peptide-like spacer to polyethylene rods to which a linear polymer of acrylic acid had been attached by radiation grafting. Overlapping octapeptides were synthesized consecutively using solid phase peptide synthesis. Following removal of the t-butyloxy carbonyl group from the final amino acid, peptides were acylated and de-protected to provide material of sufficient purity to enable ELISA testing for the specific binding of monoclonal antibody to each octapeptide [29].

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i 7 WO 88/00597 PCT/AU87/00226 12 FIGURE LEGENDS SFig. 1. Indirect immunofluorescence of fixed P. falciparum asexual blood stages reacted with MAb 8E7/55. Fluorescence micrograph shows ring trophozoite and schizont stage parasites in methanol-fixed smears of isolate FCQ-27. The antibody reacts with inclusions (arrows) inside the host cell of schizont infected erythrocytes.

Fig. 2. Indirect immunofluorescence of unfixed P. falciparum asexual blood stages reacted with MAb 8E7/55. Fluorescein staining occurs on schizont stage parasites and ruptured schizonts (rs) of isolate FCQ-27 (A and B) and FCR-3K+ Fig. 3. Immunoelectronmicrograph showing ring stage infected erythrocytes from P. falciparum labeled with MAb 8E7/55 and colloidal gold (10nm) IgG complex. The insat shows ring stage parasite surface labeling using 15nm gold particles. The bar equals 0.5 pm.

Fig. 4. Immunoelectronmicrograph of P. falciparum infected erythrocytes labeled with monoclonal antibody and colloidal gold IgG complex. Sections of schizonts were reacted with MAb 8E7/55, rabbit anit-mouse IgG and colloidal gold (10mm) labeled goat anti-rabbit antibody. Nu, nucleus; M, merozoite; PM, plasma membrane; PVM, parasitophorous vacuole membrane; R, rhoptry; RBCM, erythrocyte plasma membrane. The bar equals lym.: Fig. 5. Immunoelectronmicrograph of P. falciparum infected erythrocytes labeled with monoclonal antibody and colloidal gold IgG complex. Sections of infected erythrocytes were reacted with antibodies as described in Fig. 4 (A and schizont infected erythrocyte showing distinct separation of PVM from PM with antibody labeling restricted to the vacuole membrane; Membrane bound cleft and multimembranous vesicle showing antibody labeling. S Dense antibody labeling around membrane bound EDM free vesicle. The bar equals 0.5pm. eSHJ SU.SU1ULI '14 l i~;i -~li WO- 88/00597 PCT/AU87/00226 Fig. 6. SDS-PAGE analysis and fluorography of amino acid labeled detergent extracted proteins from FCR-3K+ and FCQ-27 strains of P. falciparum. Immunoprecipitation with MAb 8E7/55 of 14 (3 parasites labeled with [1 C] leucine; [3H] glycine acid 35 5S] methionine. Migration positions of molecular weight standards (in kD) are shown. The high molecular weight bands in c can be attributed to immunoglobulin.

Fig. 7. SDS-PAGE analysis and fluorography of fatty acid iabeled, detergent extracted proteins of P. falciparum.

Immunoprecipitation with MAb 8E7/55 of of myristic acid labeled FCR-3K+ strain parasites; myristic acid labeled FCQ-27 strain parasites. SDS-PAGE analysis and fluorography of affinity purified vacuole antigen from a FCR-3K+ strain of P. falciparum. Silver stain of purified antigens. Immunoblot of purified antigen using MAb 8E7/55 and 125-labeled goat anti-mouse IgG. The positions of molecular weight standards (in kD) are marked.

Fig. 8. Results coded for by the of scanning the predicted sequence of the polypeptide insert of Ag61, using monoclonal antibody 8E7/55.

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RESULTS

Immunofluorescence with MAb 8E7/55. The reactivity of methanol-fixed ring, trophozoite and schizont infected erythrocytes of the PNG FCQ-27 and the Gambian FCR-3K+ isolates with MAb 8E7/55 is illustrated in S Fig. 1. Both isolates reacted strongly with the monoclonal antibody at all stages, showing fluorescence around the parasite inside the infected red blood cell. The antigen recognized by the antibody was also present in inclusions located in the cytoplasm of the host cell.

Similar results were obtained in the indirect fluorescence test using unfixed parasites after release by treatment with anti-erythrocyte antibody and complement, as shown in Fig. 2 A-C. Thus fluorescence microscopic examination suggests a location of the antigen either in the parasitophorous vacuole (membrane) or in the plasma membrane of the parasite. This antigen has been designated QF116.

Immunogold labeling studies. A more precise determination of the antigen site was obtained by electronmicroscopic examination. To discriminate between location of the antigen in the parasitophorous vacuole, the parasitophorous vacuole membrane (PVM) or the plasma membrane thin sections of parasite infected erythocytes were 20 subjected to immunogold labeling. The antigen was just detectable at S" the ring stage located mainly around the parasite (Fig. while at the early and late schizont stages the antigen was found to be closely i i associated with the parasitophorous vacuole membrane with a very low background of non-specific gold labeling, occuring most frequently over the nucleus (Fig. At this stage both membranes, the PVM and the PM, were mozphologically distinctly separated (Fig. 5A and B) allowing clear identification of the localization of antigen QF116 in Sthe PVM. Dense antigen distribution was also seen in membrane bound vesicles and inclusions residing in the cytoplasm of the host cell (Fig. 5C and D) as well as close to cytoplasmic clefts. The antigen i was also localised on free membranes found associated with intact merozoites following the rupture of schizont-infected cells.

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i 1 i, ilr i Growth inhibition. Purified monoclonal antibody 8E7/55 inhibited the in vitro multiplication of FCQ-27 in a concentration dependent manner 3 as determined by H] hypoxanthine uptake. 55% inhibition was achieved at 0.5 mg ml antibody. Microscopic examination of parasites cultured in the presence of purified 8E7/55 antibody showed both fewer parasitized red blood cells and intracellular death of those parasites in red blood cells. Biosynthetic labeling with amino acids and carbohydrate. The Papua New Guinean and Gambian isolates were cultured in vitro with one or more of three amino acids, glycine and leucine from the ring to schizont stage, and methionine through the schizont stage.

Schizont infected cells were then harvested, parasites isolated and detergent soluble material immunoprecipitated with MAb 8E7/55. A single band of Mr 15 kD was detected by SDS-PAGE of immunoprecipitated material using the Gambian isolate and a Mr 19 kD band was detected using the PNG isolate (Fig. 6A and Immunoprecipitates of carbohydrates labled parasite extracts did not how significant glycosylation of the protein as judged by either galactose or glucosamine incorporation.

Metabolic labeling with fatty acid. While the molecule did not appear to be glycosylated it did show acylation by myristic acid strongly suggesting that it could be associated with membranes. When immunoprecipitating myristic acid labeled antigen from the Gambian strain parasites with MAB 8E7/55, a broad band of 15 kD as well as 25 free fatty acid and/or glycolipid could be detected in SDS-PAGE, whereas the FCQ-27 strain gave a 19 kD band (Fig. 7A).

Western blot transfer studies. After SDS-PAGE of detergent solubilized parasite extracts, proteins were electrophoretically blotted onto nitrocellulose filter paper. Immunodetection by incubation with hybridoma culture supernatant of MAb 8E7/55 and subsequently with iodinated goat-anti-mouse antibody is positive for total parasite extracts as well as for detergent soluble material giving a band at Mr 19kD. By contrast, immunoblotting using a human serum affinity-purified on the recombinant protein expressed by AG61 gave a band of Mr 23kD. Using affinity purified antigen a 15kD band was identified by blotting in the case of the FCR-3K+ strain as shown in Fig. 7B. S TITUTE SHEET 1 3.

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WO 88/00597 2/8 PCT/AU87/00226 FIGUIE 2 7 WO 88/00597 PCT/AU87/00226 Identification of epitope recognised by 8E7/55. On scanning of the predicted amino acid sequence of the iolypeptide coded for by the insert of Ag61, three octapeptides at the C-terminal end of the sequence produced strong positive ELISA signals using 8E7/55 (Fig.8).

The epitope recognised by this monoclonal antibody is contained within the sequence common to these three octapeptides, namely NLVSGP, and may be a sequence derived from the common sequence, for example VSGP.

DISCUSSION

Thus far the chemical characteristics of the antigen described in this study mark it has a membrane bound protein of small apparent molecular weight varying in size between strains. Its location can be attributed to the parasitophorous vacuole membrane of P. falciparum infected erythrocytes as well as clefts and vesicles within the cytoplasm of the host cell as shown by immunofluorescence and immunoelectron microscopy. While the molecule is apparently not glycosylated, it does contain myristic acid in its lipid moiety. The finding that a number of P. falciparum proteins are myristylated poses the question as to the purpose of this kind of protein modification.

Membrane proteins most commonly are anchored in the membrane by a transmembrane spanning hydrophobic amino acid sequence. Its replacement by a complex containing fatty acid, phosphatidyl choline, and carbohydrates has been found in the variant suface antigen (VSG) of African trypanosomes The merozoite surface coat of P.

falciparum may be constructed and function in some respects in a similar way to the trypanosome coat. The major merozoite surface glycoprotein, which contains both carboh yrate and myristic acid may show a VSG-like type of linkage of the lipid tail to the peptide chain thus enabling the parasite to shed its coat during invasion of the host cell possibly by means of an endogenous phopholipase. In contrast, this parasitophorous vacuole membrane antigen associated .ith a membrane inside the host cell, evidently lacks the carbohydrate moiety and its fatty acid residue may be bound to the peptide backbone in an ester or amide linkage as described for tumour virus proteins I 1.

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TrcJ i^^l 1 WO 88/6--:-9 PCT/AU7/00226: 17 The scan of the Ag61 insert sequence shows that antibody 8E7/55 can bind to an epitope coded for by this sequence. The Ag61 sequence from FCQ-27/PNG is almost identical to the Ag5.1 sequence reported by Hope et.al. [34] from the K1 isolate of P.falciparum, and on the basis of the epitope scanning Ag5.1 would also contain this epitope. However, the predominant aat5ogn according to the present invention is smaller than that reported for the native antigen in FCQ-27/PNG corresponding to the Ag61 clone sequence (19kD vs. 23kD).

This difference in size is real since immunoblotting of parasite extracts with human antibodies purified on the Ag61 clone product gives the reported size of 23kD in the system described herein. The size of the predominant antigen recognised by 8E7/55 in the FCR-3K+ isolate is even smaller than that in the FCQ-27/PNG isolate, and is much smaller than that of the Ag5.1 antigen reported for any isolate.

Taken together, these data show that although the 8E7/55 monoclonal antibody could theoretically bind to the Ag5.1 protein, the dominant antigen (designated QF116 herein) recognised in parasite extracts is distinctly smaller.

The results from the growth inhibition experiments suggest that antibody 8E7/55 may kill parasites by two mechanisms. Firstly, by aggregating membranes released during rupture of the schizontinfected red blood cells thereby blocking invasion of merozoites.

Secondly, the antibody has an unexpected direct effect on intraerythrocytic parasites resulting in their death. A plausible mechanism isthe interferencef trafficking of membranous structures from the parasitophorous vacuole to the surface, and vice versa.

Whether the growth inhibition is caused by binding of 8E7/55 monoclonal antibody to Ag5.1, iQF116 or to some other as yet undescribed antigen, the investigation of the specificity of this monoclonal antibody sh-ws that such inhibition will depend upon the presence of the sequence NLVSGP, or some closely related antigenically active sequence.

ii 1 1 i i 1 i I ;I j 1/ "a T:i ^ij WO 88/00597 PCT/A U87/00226 18 References 1. Nardin, Nussenzweig, Nussenzweig, Collins, Harinasuta, Tapchaisri, P. and Chomcharn, Y.

(1982) Circuznsporosoite proteins of human malaria parasites Plasmodium falcivaru and Plasmodium vivax. J. Exp. Med. 156, 20-30., 2. 'Heidrich, Strych, W. and Mrema, J.E.K. (1983) Identification of surface and internal antigens from spontaneously released Plasmodium falciparum merozoites by radio-iodination and metabolic labelling. Z. Parasiten k. 69, 715-725., 3, Freeman, P.R. and Holder, A.A. (1983) Surface antigens of malaria merozoites. A high molecular weight precursor is processed to an 83,000 mol wt form expressed on! the surface of Plasmodium falciparun merozoites J. Exp. Med. 158, 1647-1653.

4. Brown, Culvenor, Creawther, Bianco, A.E., t Coppel, Saint, Stahl, Kemp, D.J. and'Anders R.F. (1985) Localisation of the ring-infected erythrocyte surface antigen (RESA) of Plasmodiumn falciparun in merozoites and ring infected erythrocytes. J. Exp. Med. 162. 774-779.

McLaren, Bannister, Trigg, P. and Butcher, G.

(1979) Freeze fracture studies on the interaction between the malaria parasite and host erythrocyte in Plasmodijii knowlesi Vinfections. Parasitology 79, 125-139.

6. Sherman, J.W. (1985) Membrane structure and function of f malaria parasites and the infected erythrocyte. Parasitology 91, 609-645.

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WO 88/00597 PCT/AU87/00226 19 7. Hope. Hall, Simmons, Hyde, J.E. and Scaife.

J.G. (1984). Evidence for immunological cross.-reaction between sporozoites and blood stages of a human malaria parasite. Nature (London) 308, 191-193.

8. Saul, Myler, Schofield, L. and Kidson, C. (1984). A high molecular weight antigen in Plasmodium falciparun recognised by inhibitory monoclonal antibodies. Parasite Immunology 6, 39-50.

9. Saul, Cooper, Ingram, Anders, R.F. and Brown, G.V. (1985). Invasion of erythrocytes in vivo by Plasmodium falciparum can be inhibited by"'monoclonal antibody against an S-antigen. Parasite Immunology 7, 587-593.

Chen, Lamnont, G. Elliot, T. Kidson, Brown, G., Mitchell, Stace, J. and Alpers, M. (1980) Plasmnodium falciparum strains from Papua New Guinea: culture characteristics an drug sensitivity. Southeast Asian J.

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Parasitol. 65, 418-420.

12. Saul, Myler, Elliot, T. and Kidson, C. (1982) Purification of mature schizonts of Plasmodiumn falciparum on colloidal silica gradients. Bull. WHO 60, 755-759.

13. Anders, Brown, GiV. and Edwards, A. (1983) Characterisation of an S-antigen synthesized by several i isolates of Plasmodiumi falciparum. Proc. Natl. Acad. Sci.

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4 I WO0 88/00597 PCT/A U87/00226 Schofield, Saul, Myler, P. and Kidson. C. (1982) Antigenic differences between isolates of Plasmodiun falciparun demonstrated by monoclonal antibodies. Infect.

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Haldar, Ferguson, MA.J. and Cross, G.A.M. (1985) Acylation of a Plasmodiumn falciparui merozoite surface antigen via sn-i, 2-diacylgycerol. J. Eiol. Chem. 260, 4972-4974.

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(1985) An N-terminal peptide from p 60 sr can direct ~~ryristylation and plasma membrane localisation when fused to heterologons proteins. Nature 314, 374-376.

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q 0O88/00597 PCT/ AU87/00226 '4 WO 88/00597 PCT/A U87/00226 Aikawa, Miller, L.H. and Rabbage, J. (1975) Careola Vesicle complexes in the plasmalemma of erythrocytes infected by Plasmodium vivax and P. cynomolgi Am. J. Pathol. 79, 285-300.

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Claims (12)

1. A small molecular weight antigen of the asexual blood stages of Plasmodium falciparum which is characterised by: having an apparent molecular weight in the range of app ioximately 15 kD and 19 kD; (ii) not showing significant glycosylation by galactose or glucosamine labelling, but being acylated by myristic acid; (iii) being associated with the parasitophorous vacuole membrane and with inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell; and (iv) being recognised by monoclonal antibodies against the asexual blood stages of P.falciparum which inhibit parasite growth in vitro; or an antigenic fragment thereof.

2. An antigen according to claim 1, which is antigen QF116 described herein; or an antigenic fragment thereof.

3. A hybrid cell line which produces an antibody which is specific for the small molecular weight antigen according to claim 1.

4. A hybrid cell line according to claim 3 which is the cell line deposited under No. 87071009. An antibody produced by a hybrid cell line according to claim C1:1

6. An antibody according to claim 5, which is the monoclonal antibody 8E7/55 described herein.

7. An antigen of Plasmodium falciparum which is characterised by the inclusion of the amino acid sequence Asn-Leu-Val-Ser-Glu-Pro (NLVSGP), or anki or an :II. 31 j- i i j_ ~~-lrj IL4 0i CIIP--( ^ar 2 WO 88/00597 :PCT/AU87/0226 antigenic fragment thereof.

8. A method of inhibiting the development of asexual blood stages of Plasmodium falciparum, which method comprises exposing the blood stages of the parasite to an antibody according to claim

9. A method for passively immunising a host against Plasmodium falciparum, which method comprises administering to the host an antibody according to claim A passive vaccine composition comprising an antibody according to claim 5, and a pharmaceutically acceptable carrier or diluent.

11. A composition according to claim 10, wherein the antibody is monoclonal antibody 8E7/55.

12. A method for actively immunising a host against Plasmodium falciparum, which method comprises administering to the host an antigen according to claim 1 or claim 7, or an antigenic fragment thereof.

13. A vaccine composition comprising an antigen according to claim 1 or claim 7, or an antigenic fragment thereof, and a pharmaceutically acceptable carrier or diluent.

14. A composition according to claim 13, further comprisir' "an adjuvant. A composition according to claim 13, wherein antigen QF116, or an antigenic fragment thereof. the antigen is rI i i r I i :I r :L r WO 88/00597 WO 8800597PCT/AU87/00226 1.6. A synthetic peptide comprising an antigenic fragment having an amino acid sequence which comprises or includes the seguence Asn-Leu-Val-Se-',.u?ro (NLVSGP), or an~gni&1 aet. Jz~ ~0 ~s 1.. V .1 I I.